Combination therapy, restricted to reverse transcriptase and protease inhibitors, has dramatically improved outcomes of HIV-infected patients. In spite of the initial success, the increased prevalence of HIV strains resistant to multiple FDA-approved antiretroviral drugs mandates the design of agents directed to new therapeutic targets in HIV, such as the envelope protein. HIV infection is initiated by binding of the viral envelope to CD4 at the cell membrane. This induces a conformational change in gp120 to reveal cryptic epitopes important in viral entry. In particular, interaction of gp120 V3 loop with chemokine receptors occurs after gp120 cenformational changes, when the V3 loop becomes more exposed. The V3 loop is a critical determinant for HIV tropism via its interaction with the chemokine receptor CCR5 or CXCR4, and anti-V3 loop monoclonal antibodies can be neutralizing. The VIN2 loop may be an important determinant in shielding the V3 loop and other regions essential for binding to the appropriate HIV co-receptor. Since the VIN2 and V3 domains contain critical determinants for coreceptor interactions, these loops are suitable targets for the design of small molecule inhibitors. Our goal is to identify high-affinity small peptides that target VIN2 and V3 regions that may inhibit viral entry. We plan to generate hybrid proteins containing these loops for use as targets in the selection of specific high affinity peptides from combinatorial libraries. In order to obtain peptides specific to these loops, we will generate constructs that constrain gp120 V3 or VlN2 loops grafted into regions of murine leukemia virus (MLV) envelope protein or Staphylococcal nuclease (SN), which are well-characterized scaffold proteins that can tolerate large insertions while maintaining their native structure. Phage display will be used to select high affinity peptides directed to these loops from combinatorial libraries. The affinity of phagederived peptide(s) for the VlN2 and V3 constructs will then be assayed. There is high probability that small molecules binding to these gp120 regions will disrupt viral entry, and may be used for the development of new antiretroviral drugs. Currently, peptides targeting the envelope structure have demonstrated clinical efficacy in patients resistant to other antiretroviral therapies. Since our peptides will be targeting viral structures, we expect them to have low toxicity, and no cross-resistance with the current antiretroviral drugs.